Increase in methicillin-resistant Staphylococcus spp. colonization among pregnant individuals during COVID-19 pandemic

Methicillin-resistant Staphylococcus (MRS) has been associated with neonatal infections, with colonization of the anovaginal tract being the main source of vertical transmission. The COVID-19 pandemic has altered the frequency of antibiotic usage, potentially contributing to changes in the dynamics of bacterial agents colonizing humans. Here we determined MRS colonization rates among pregnant individuals attending a single maternity in Rio de Janeiro, Brazil before (January 2019–March 2020) and during (May 2020–March 2021) the COVID-19 pandemic. Anovaginal samples (n = 806 [521 samples before and 285 during the pandemic]) were streaked onto chromogenic media. Colonies were identified by MALDI-TOF MS. Detection of mecA gene and SCCmec typing were assessed by PCR and antimicrobial susceptibility testing was done according to CLSI guidelines. After the onset of the pandemic, MRS colonization rates increased significantly (p < 0.05) from 8.6% (45) to 54.7% (156). Overall, 215 (26.6%) MRS isolates were detected, of which S. haemolyticus was the most prevalent species (MRSH, 84.2%; 181 isolates). SCCmec type V was the most frequent among MRS (63.3%; 136), and 31.6% (68) of MRS strains had a non-typeable SCCmec, due to new combinations of ccr and mecA complexes. Among MRS strains, 41.9% (90) were resistant to at least 3 different classes of antimicrobial agents, and 60% (54) of them were S. haemolyticus harboring SCCmec V. MRS colonization rates and the emergence of multidrug-resistant variants detected in this study indicate the need for continuing surveillance of this important pathogen within maternal and child populations.

Staphylococci are abundant in the human microbiota, and the main sites of colonization are skin and mucous membranes 1 .Staphylococcus aureus is the most clinically relevant coagulase-positive species and one of the leading causes of community-acquired and nosocomial infections 2 .Coagulase-negative staphylococci (CoNS) cover a broader group of species, which can act as important opportunistic pathogens, mainly associated with immunocompromised patients, long periods of hospitalization, and medical procedures using indwelling devices 1 .In this scenario, CoNS stand out as a common cause of neonatal infections, mainly associated with the occurrence of neonatal sepsis, which is a frequent type of infection in newborns 3,4 .Among CoNS, S. haemolyticus has gained special attention, becoming the second most common CoNS isolated from nosocomial infections 5 , and predominantly found in neonatal intensive care units 3,4,6 .
Methicillin-resistant Staphylococcus (MRS) are of major importance in clinical settings due to its high rates of antibiotic resistance 1,2 .Methicillin-resistance gene mecA is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec) and encodes a penicillin-binding protein with low affinity to β-lactams, called PBP-2a (or PBP-2'), which results in broad-spectrum resistance to this class of antimicrobial agents 2 .

Discussion
Staphylococcus spp., highlighting the methicillin-resistant variants, are a main cause of nosocomial and community-acquired bacterial infections worldwide 5,13,14 , being emergingly associated with neonatal infections 15,16 .However, in resource limited countries, such as Brazil, where clinical and epidemiological surveillance practices during perinatal care are still scarce, these pathogens remain neglected.To our knowledge, this is the first study that evaluates the prevalence of MRS strains in anovaginal specimens during pregnancy in Brazil.
The most frequent MR-CoNS species in this study was S. haemolyticus, with a colonization rate of 84.2%.This species has a well-established role in harboring and transferring genetic material to other staphylococci, both CoNS and coagulase-positive staphylococci (CoPS) 17,18 .Hence, the presence of MRSH strains in the human microbiota can promote the dispersion of antimicrobial resistance determinants to other microorganisms colonizing the same niche.For MRSA, the anovaginal colonization rate (2.3%) found in this study was similar to the rate reported in other studies 19,20 .
Empiric treatment for COVID-19 patients was a common practice in many hospital settings worldwide, which included different antimicrobial agents such as macrolides and carbapenems 21,22 .In many countries, azithromycin was the most prescribed drug during this period 23 , including Brazil, where increased consumption of azithromycin was reported during the pandemic 24 .The abuse of antimicrobial agents is a well-known risk factor for the emergence of MDR pathogens 10,21,22 .Therefore, the significant increase in MRS colonization during the COVID-19 pandemic could be reflecting the impact of antibiotic overuse during this period.
Similar to our findings, other studies also reported high rates of non-susceptibility to macrolides in MRS, above 70% 25,26 , and non-susceptibility to tetracycline being considerably lower, between 19 and 26% 27,28 .For lincosamides, quinolones and sulfamethoxazole/trimethoprim, the non-susceptibility rates were below 25%, which are lower than other rates reported in the literature, all higher than 40% 13,25 .However, considering the overall antimicrobial susceptibility pattern, a great proportion of the tested strains were MDR.In this context, once again S. haemolyticus stands out, having the highest proportion (47%) of MDR strains among all MR-CoNS species identified in this study.This data highlights S. haemolyticus as an important CoNS associated with MDR phenotype, which has been pointed out in the literature in the last decades 5,13,27,29 , with reported MDR prevalence as high as 75.3% among this species 26 .
In this study, iMLS B was a common phenotype among the tested strains, particularly those recovered during the pandemic.Since these strains may not respond well to clindamycin, potentially leading to treatment failure 30 , this finding emphasizes the importance of implementing the D-zone test in routine disk diffusion assays, especially in the clinical context, to ensure the detection of both constitutive and inducible resistance to clindamycin.
SCCmec typing is a common and well characterized typing method used in S. aureus strains.Most MRSA strains (60%) in this study harbored SCCmec IV, which has been reported as the most prevalent among MRSA strains associated with community-acquired infections in Brazil 14,31 .However, considering the possibility of a large variability of SCCmec elements in CoNS, applying SCCmec typing methods for this group becomes challenging, mostly because such methods were established for S. aureus.MRS-CoNS can either present new combinations of mec and ccr complex genes or present more than one mec and ccr complex in a single strain 29,[32][33][34] .In the present study, we detected a considerable amount of NT strains in MRS strains (31.6%), being as high as of previous reports, which ranged from 13.2 to > 50% 33,35,36 , with several strains having more than one ccr gene complex with a single mec gene complex.These findings were also reported by other groups, 29,[32][33][34]37 with the same combinations of ccr and mec gene complexes also found in our study, such as a ccr type 2 and ccr type 5 with mecA class C, and ccr type 1 and ccr type 5 with mecA class A.
As limitations of this study, we recognize that collecting anovaginal specimens from pregnant individuals at maternities from different locations could contribute to better comprehend and establish the epidemiological scenario and characterization of MRS colonization in this population, in Rio de Janeiro city.Moreover, the lack of data regarding methicillin-susceptible Staphylococcus colonization in our study sample may have impacted the comparison and analysis of MRS colonization dynamics among the whole Staphylococcus population colonizing the anovaginal site.
The increase in MRS colonization rate during the pandemic and the large number of strains with MDR phenotype observed in this study contribute to highlight the potential impact of the COVID-19 pandemic in the dynamics of bacterial infections, especially those associated with antimicrobial resistance.Thus, our findings point out to the need for developing and improving surveillance and public health policies for this bacterial group, mainly in special populations such as pregnant individuals and neonates, aiming to contribute to maternal and child health.

Clinical samples
This is a descriptive study on anovaginal colonization by methicillin-resistant Staphylococcus spp.during pregnancy in Rio de Janeiro, Brazil.Anovaginal specimens were collected from all pregnant individuals attending the ME-UFRJ, between January 2019 and March 2021, being separated in two scenarios according to the date of collection: before COVID-19 was declared a pandemic by the World Health Organization (January 2019-March 2020) and during the COVID-19 pandemic (May 2020-March 2021).Although clinical samples were collected constantly during the period evaluated, no samples were collected on April 2020 due to the COVID-19 lockdown.The ME-UFRJ is a specialized teaching maternity, providing outpatient care to individuals with high-risk pregnancies.The maternity is located in a major metropolitan area of Rio de Janeiro State, the city of Rio de Janeiro, in the Southeastern region of Brazil.
The specimens were obtained between the 35th and 37th gestational week during routine antenatal care, using the combined swab method according to previous guidelines 39 .All participants signed a consent form upon the sample collection.This project was approved by the Research Ethics Committee of UFRJ (Number: 43389321.9.0000.5257).The clinical samples were placed in cryogenic tubes containing STGG media (skim milk, tryptone, glucose and glycerol) until processing.

Samples processing
Anovaginal specimens were streaked onto chromogenic media CHROMagar™ MRSA (CHROMagar, Paris, France) and incubated for 24 h at 37 °C.After this period, one representative colony of each morphotype was selected, based on colony size and color, and identified using MALDI-TOF MS (Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry; Bruker Microflex LT, Bruker Daltonics, Bremen, Germany).Following identification, strains were stored in skim-milk with glycerol (10%) at − 20 °C.
Methicillin-resistant strains had their DNA extracted for mecA gene detection and SCCmec typing, using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), according to the manufacturer's instructions.
Detection of the mecA gene was accessed in accordance with Del Vecchio et al. 41 .For determining the SCCmec types, multiplex PCR reactions were performed as presented by Kondo et al. 42 .

Statistical analysis
Statistical analyses were performed using GraphPad Prism version 8.0.2 (GraphPad Software, Boston, MA, USA).Chi-square and Fisher's test were applied and p-value less than 0.05 was considered significant.

Institutional review board
The study was conducted in accordance with the Declaration of Helsinki and approved by the local Ethics Committee of University Hospital Clementino Fraga Filho of the Federal University of Rio de Janeiro (UFRJ) (protocol code 43389321.9.0000.5257and date of approval January 2020).

Table 1 .
Distribution of antimicrobial susceptibility rates and SCCmec types among the five most prevalent species found in this study.MDR: multidrug resistance, NT: non-typeable.